Overexpression of the RieskeFeS Protein IncreasesElectron Transport Rates and Biomass Yield1[CC-BY]

Andrew J. Simkin, Lorna McAusland,2 Tracy Lawson, and Christine A. Raines3

School of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom

ORCID IDs: 0000-0001-5056-1306 (A.J.S.); 0000-0002-5908-1939 (L.M.); 0000-0002-4073-7221 (T.L.); 0000-0001-7997-7823 (C.A.R.).

In this study, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing the Rieske FeS protein (PetC), acomponent of the cytochrome b6f (cyt b6f) complex. Increasing the levels of this protein resulted in concomitant increases in the levels ofcyt f (PetA) and cyt b6 (PetB), core proteins of the cyt b6f complex. Interestingly, an increase in the levels of proteins in both thephotosystem I (PSI) and PSII complexes also was seen in the Rieske FeS overexpression plants. Although the mechanisms leading tothese changes remain to be identified, the transgenic plants presented here provide novel tools to explore this. Importantly,overexpression of the Rieske FeS protein resulted in substantial and significant impacts on the quantum efficiency of PSI and PSII,electron transport, biomass, and seed yield in Arabidopsis plants. These results demonstrate the potential for manipulating electrontransport processes to increase crop productivity.

Increasing food and fuel demands by the growingworld population has led to the need to develop higheryielding crop varieties (Fischer and Edmeades, 2010; Rayet al., 2012). Transgenic studies, modeling approaches,and theoretical considerations provide evidence thatincreasing photosynthetic capacity is a viable route toincrease the yield of crop plants (Zhu et al., 2010; Raines,2011; Long et al., 2015; von Caemmerer and Furbank,2016). There is now a growing body of experimentalevidence showing that increasing the levels of photo-synthetic enzymes in carbon metabolism results in in-creased photosynthesis and plant biomass (Miyagawaet al., 2001; Lefebvre et al., 2005; Raines, 2006, 2011;Rosenthal et al., 2011; Uematsu et al., 2012; Simkin et al.,2015, 2017; Driever et al., 2017). In addition, the manip-ulation of photosynthetic electron transport by the in-troduction of the algal cytochrome c6 protein has beenshown to improve the efficiency of photosynthesis andto stimulate plant growth in low light (Chida et al., 2007).

One endogenous target identified formanipulation is thecytochrome b6f (cyt b6f) complex, which is located in thethylakoid membrane and functions in both linear andcyclic electron transport, providing ATP and NADPHfor photosynthetic carbon fixation. Initially, cyt b6f in-hibitors (Kirchhoff et al., 2000), and later, transgenicantisense studies suppressing the accumulation of theRieske FeS protein (PetC), a component of the cyt b6fcomplex, demonstrated that the activity of the cyt b6fcomplex is a key determinant of the rate of electrontransport (Price et al., 1995, 1998; Anderson et al., 1997;Ruuska et al., 2000; Yamori et al., 2011a, 2011b).

The finding that the cyt b6f complex is a potentiallimiting step in the electron transport chain suggeststhat, by increasing the activity of this complex, it may bepossible to increase the rate of photosynthesis. However,questions have been raised about the feasibility of ma-nipulating this multiprotein, membrane-located complex,given that it is composed of eight different subunits, sixbeing encoded in the chloroplast genome (PetA [cyt f],PetB [cyt b6], PetD, PetG, PetL, and PetN) and two in thenucleus (PetC [Rieske FeS] and PetM; Willey and Gray,1988; Anderson, 1992; Knight et al., 2002; Cramer andZhang, 2006; Cramer et al., 2006; Baniulis et al., 2009;Schöttler et al., 2015). Furthermore, this protein complexfunctions as a dimer,with the transmembrane domains ofboth the Rieske FeS and cyt b6 proteins being implicateddirectly in the monomer-monomer interaction and sta-bility of the complex and the petD gene product func-tioning as a scaffold (Hager et al., 1999;Cramer et al., 2006;Schwenkert et al., 2007; Hojka et al., 2014). Essential rolesin the assembly and stability of the cyt b6f complex alsohave been shown for the PetG, PetN, and PetM subunits,and aminor role in stabilitywas assigned to the PetL geneproduct (Bruce and Malkin, 1991; Kuras and Wollman,1994; Hager et al., 1999;Monde et al., 2000; Schöttler et al.,2007; Schwenkert et al., 2007; Hojka et al., 2014).

1 This work was supported by the Biotechnology and BiologicalSciences Research Council (grant no. BB/J004138/1 awarded toC.A.R. and T.L.).

2 Current address: Division of Crop and Plant Science, School ofBiosciences, University of Nottingham, Nottingham LE12 5RD, UK.

3 Address correspondence to rainc@essex.ac.uk.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Christine A. Raines (rainc@essex.ac.uk).

A.J.S. generated transgenic plants, performed molecular and bio-chemical experiments, and carried out plant phenotypic and growthanalyses; A.J.S. and L.M. performed gas-exchange measurement onArabidopsis; A.J.S. and L.M. carried out data analysis on their respec-tive contributions; C.A.R. and T.L. designed and supervised the re-search; C.A.R. and A.J.S. wrote the article with input from T.L.

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Notwithstanding both the genetic and structural com-plexity of the cyt b6f complex, it has been shown pre-viously that it is possible to manipulate the levels ofthe cyt b6f complex by down-regulation of the ex-pression of the Rieske FeS protein (Price et al., 1998;Yamori et al., 2011b). It also has been shown that theRieske FeS protein is one of the subunits required forthe successful assembly of the cyt b6f complex (Miles,1982; Metz et al., 1983; Barkan et al., 1986; Andersonet al., 1997). Based on these results, we reasoned thatoverexpression of the Rieske FeS protein could be afeasible approach to take in order to increase theelectron flow through the cyt b6f complex. In this ar-ticle, we report on the production of Arabidopsis(Arabidopsis thaliana) with increased levels of the to-bacco (Nicotiana tabacum) Rieske FeS protein, and weshow that this manipulation resulted in increases inphotosynthetic electron transport, CO2 assimilation,and yield. This work provides evidence that the pro-cess of electron transport is a potential route for theimprovement of plant productivity.

RESULTS

Production and Selection of Rieske FeSOverexpression Transformants

The full-length tobacco Rieske FeS coding sequence(X64353) from the cyt b6f complex was used to generatean overexpression construct B2-NtRi (SupplementalFig. S1). Following floral dipping, transgenic Arabi-dopsis plants were selected on both kanamycin- andhygromycin-containing medium (Nakagawa et al., 2007),and plants expressing the integrated transgenes wereidentified using reverse transcriptase-PCR (data notshown). Proteins were extracted from leaves of the T1progeny, allowing the identification of three lines withincreased levels of the Rieske FeS protein (PetC;Supplemental Fig. S2A). Immunoblot analysis of T3progeny of lines 9, 10, and 11 showed them to havehigher levels of the Rieske FeS protein when comparedwith the wild type (Fig. 1; Supplemental Fig. S2B). Theoverexpression of the Rieske FeS protein (hereafterreferred to as Rieske FeS ox) resulted in a concomitant

Figure 1. Immunoblot analysis of leaf proteins of wild-type (WT) and Rieske FeS ox plants. Protein extracts were from leaf discstaken from two leaves per plant from three independent lines (9, 10, and 11) and separated on a 12% acrylamide gel, trans-ferred to membranes, and probed with relevant antibodies. The cyt b6f complex subunits PSI and PSII, ATP synthase d-subunit(AtpD), Calvin-Benson cycle (CBC) proteins, and photorespiratory GDC-H protein were probed. A, Protein (6 mg) was loadedfor all antibodies except for FBPA (3 mg) and Rubisco (1 mg). B, Proteins were loaded containing 0.63 to 10 mg of protein.C, Proteins were quantified using a Fusion FX Vilber Lourmat imager (Peqlab), as described previously (Vialet-Chabrand et al.,2017), and are presented as relative protein contents compared with the wild type.

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increase in both cyt f (PetA) and cyt b6 (PetB; Fig. 1A).An increase in the level of the PSI type I chlorophyll a/b-binding protein (LhcaI) and an increase in the coreprotein of PSI (PsaA) also were observed. Furthermore,the D1 (PsbA) and D2 (PsbD) proteins, which form thereaction center of PSII, also were shown to be elevatedin Rieske FeS ox lines. Finally, an increase in the ATPsynthase d-subunit (AtpD) also was observed in RieskeFeS ox lines (Fig. 1A). In contrast, no notable differencesin protein levels for the chloroplastic FBP aldolase(FBPA), the mitochondrial Gly decarboxylase-H pro-tein (GDC-H), or the Rubisco large subunit were ob-served (Fig. 1A). A quantitative estimate of the changesin protein levels was determined from the immunoblotsof leaf extracts isolated from two to three independentplants per line. An example is shown in Figure 1B.These results showed a 2- to 2.5-fold increase in theRieske FeS protein relative to wild-type plants, and asimilar increase also was observed for cyt f, cyt b6,Lhca1, D2, and PsaA (Fig. 1C). No increase in thestromal FBPA protein was evident.

Chlorophyll Fluorescence Imaging Reveals IncreasedPhotosynthetic Efficiency in Young Rieske FeSox Seedlings

In order to explore the impact of increased levels ofthe Rieske FeS protein on photosynthesis, the quantumefficiency of PSII (Fq9/Fm9) was analyzed using chlo-rophyll a fluorescence imaging (Baker, 2008; Murchieand Lawson, 2013). A small increase in Fq9/Fm9 wasfound in the Rieske FeS ox plants at irradiances of310 and 600 mmol m22 s21 (Fig. 2). Leaf area, generatedfrom these images, was significantly larger in all RieskeFeS ox lines compared with the wild type (Fig. 2C), butno significant difference in leaf thickness was observedbetween the leaves of Rieske FeS lines 9 and 11 and thewild-type plants (Supplemental Table S1).

Photosynthetic CO2 Assimilation and Electron TransportRates Are Increased in the Rieske FeS ox Plants

The impact of overexpression of the Rieske FeS pro-tein on the rate of photosynthesis in mature plants wasinvestigated using combined gas-exchange and chlo-rophyll fluorescence analyses. Both the light-saturatedrate of CO2 fixation and the relative light-saturated rateof electron transport (ETRII) were increased in theRieske FeS ox lines compared with the wild type whenmeasured at 2% [O2] (Fig. 3, A and B; Table I). Addi-tionally, the light-saturated rate of CO2 assimilation atambient [CO2] also was increased when measured at21% [O2] (Supplemental Fig. S3). No significant differ-ence in leaf absorbance between the Rieske FeS ox andwild-type plants was found (Supplemental Table S1).

In plants grown at a light level of 130 mmol m22 s21,no difference in the light- or CO2-saturated rate of CO2assimilation (Amax) was found. In contrast, in a second

group of plants grown at 280 mmol m22 s21, Amax wasgreater in the Rieske FeS ox lines 9 and 11 relative to thewild type (Fig. 3C; Table I). Further analysis of theA/Cicurves revealed that maximum electron transport ratewas significantly greater in the Rieske FeS ox plantswhen compared with the wild type (Table II), but nosignificant difference in the maximum rate of Rubisco(data not shown) was observed.

The Quantum Efficiency of PSI and PSII Was Increased inthe Rieske FeS ox Plants

To further explore the influence of increases in theRieskeFeS protein on PSII and PSI photochemistry, dark-light

Figure 2. Determination of photosynthetic efficiency and leaf area inRieske FeS ox seedlings using chlorophyll fluorescence imaging. Fq9/Fm9 at310mmolm22 s21 (A andD) and 600mmolm22 s21 (B) and leaf area at thetime of analysis (C). The data were obtained using four to six individualplants from each line comparedwith the wild type (five plants). Significantdifferences (P , 0.05) are represented as uppercase letters indicating dif-ferences between lines. Lowercase italic letters indicate lines that are justbelow significance (P . 0.05 and , 0.1). Error bars represent SE. D, Wildtype (WT) and Rieske FeS ox plants were grown in controlled-environmentconditions with a light intensity of 130 mmol m22 s21, 8-h-light/16-h-darkcycle, and chlorophyll fluorescence imagingwas used to determine Fq9/Fm9at two light intensities 14 d after planting (DAP).

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induction responses were determined in the wild type andRieske FeS ox (lines 11 and 10) using simultaneousmeasurements of P700 oxidation state and PSII effi-ciency. These results showed that the quantum yieldsof both PSI and PSII were increased in the Rieske FeSox plants compared with the wild type and that thefraction of PSII centers that were open (qL) also wasincreased, while the level of Qa reduction (12 qp) waslower in leaves of 27-d-old plants from line 11 (Fig. 4).non-photochemical quenching (NPQ) levels also wereshown to be lower in the Rieske FeS ox plants, togetherwith a reduction in stress-induced limitation of NPQ(qN) when compared with wild-type plants (Fig. 4).Similar results were obtained for both lines 10 and11 when plants were analyzed later in development(34 DAP; Supplemental Fig. S4). The increases in thequantum yields of PSI and PSII observed here wereaccompanied by corresponding increases in electrontransport rates (ETRI and ETRII; Supplemental Fig. S5,A and D); however, a bigger increase in ETRII relativeto ETRI was observed, demonstrated by the wild typehaving a higher ETRI-ETRII ratio (Supplemental Fig.S5, C and D).

Growth, Vegetative Biomass, and Seed Yield Are Increasedin the Rieske FeS ox Plants

The leaf area of the Rieske FeS ox lines was signifi-cantly greater than that of the wild type as early as10 DAP in soil, and by 18 d it was 40% to 114% larger(Fig. 5). Destructive harvest at day 25 showed that thisincrease in leaf area translated to an increase in shootbiomass of between 29% and 72% determined as dryweight (Fig. 5C). To determine the impact of increasedRieske FeS protein on seed yield and final shoot bio-mass, a second group of plants was grown in the sameconditions as described in Figure 6. Interestingly, at38 DAP, 40% of the Rieske FeS ox plants had flowered,in contrast to 22% in the wild-type plants (Fig. 6A).Following seed set (52 DAP), both vegetative biomass(Fig. 6B) and seed yield (Fig. 6C) were determined, andalthough a significant increase in biomass was ob-served in all of the Rieske FeS ox plants, a statisticallysignificant increase in seed yield was evident only inline 11.

Pigment Content Was Altered in the Rieske FeS ox Plants

The pigment composition of the leaves of the RieskeFeS ox and wild-type plants was determined. Increasesin the levels of chlorophyll a and b (14%–29%) wereobserved in the Rieske FeS ox compared with wild-type

Figure 3. Photosynthetic responses of the Rieske FeS ox plants. A and B,Determination of photosynthetic capacity (A) and electron transportrates (B) in transgenic plants at 2% [O2]. Wild-type (WT) and transgenicplants were grown in controlled-environment conditions with a lightintensity 130 mmol m22 s21 and an 8-h-light/16-h-dark cycle for4 weeks. C, Photosynthetic carbon fixation rate (A) was determined as afunction of increasing CO2 concentrations (A/Ci) at saturating light

levels (1,000 mmol m22 s21). Wild-type and transgenic plants weregrown in controlled-environment conditions at a light intensity280 mmol m22 s21 and a 12-h-light/12-h-dark cycle for 4 weeks. Errorbars represent SE.

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plants (Fig. 7). Plants with increased Rieske FeS proteinalso were found to have a small increase in the chlo-rophyll a/b ratio, from 2.96 to 3.12. These increaseswere accompanied by increases in the carotenoids,neoxanthin (+38%), violaxanthin (+59%), lutein (+75%),and b-carotene (+169%). No detectable change in thelevel of zeaxanthin was evident in the Rieske FeS oxplants. The increases in both total chlorophyll and totalcarotenoids also led to a significant decrease in thechlorophyll-to-carotenoid ratio, from 3.6 to 2.4 (Fig. 7).

DISCUSSION

In recent years, increasing the rate of photosyntheticcarbon assimilation has been identified as a target forimprovement to increase yield. Evidence to support thishas come from the theory and modeling of the photo-synthetic process, growth of plants in elevated CO2, andtransgenic manipulation (Zhu et al., 2010). It wasshown previously in antisense studies that reducing thelevels of the Rieske FeS protein resulted in a reductionin levels of the cyt b6f complex, a decrease in photo-synthetic electron transport, and, in rice (Oryza sativa), adecrease in both biomass and seed yield (Price et al.,1998; Yamori et al., 2016). These findings identified thecyt b6f complex as a limiting step in electron transportand would suggest that overexpression of the RieskeFeS protein may be a feasible route to increase photo-synthesis and yield. In this study, we show that over-expression of the Rieske FeS protein inArabidopsis resultsin increases in photosynthesis, vegetative biomass, andseed yield.

Increased Levels of the Rieske FeS Protein IncreasedPhotosynthetic Electron Transport, CO2 Assimilation,and Biomass

Using chlorophyll fluorescence imaging, we haveshown that overexpression of the Rieske FeS proteinresulted in increases in photosynthesis and growth, whichare evident from the early stages of seedling development.These observed increases in Fq9/Fm9 represent an early

indication that the potential quantum yield of PSII pho-tochemistry had been elevated in Rieske FeS ox lines(Genty et al., 1989, 1992; Baker et al., 2007). This earlystimulation is maintained into maturity, and increases inthe light-saturated rate of CO2 assimilation and electrontransport rates were evident in the Rieske FeS ox plants.Our data also showed that quantum yields of both PSIIandPSIwere increased and that the fraction of PSII centersavailable for photochemistry was increased, as indicatedby an increase in qL and a lower 1 2 qp (Baker andOxborough, 2004; Kramer et al., 2004; Baker et al.,2007). These results are consistent with what would bepredicted from results obtained from the Rieske FeSantisense studies where ETR was reduced (Price et al.,1998; Ruuska et al., 2000; Yamori et al., 2011b). However,the impact of overexpression of the Rieske FeS proteinwas clearly not restricted to increasing the activity of thecyt b6/f complex but resulted in changes in PSI, PSII, andthe ATPase complex, which led to an increase in electronflow through the entire electron transport chain. Anal-ysis of the ratio of PSI to PSII electron transport showedthat, relative towild-type plants, the Rieske ox lines havehigher rates of PSII ETR relative to PSI, which may berelated to cyclic electron flow, differences in energydistribution, and/or the PSI-PSII ratio (Kono et al., 2014).

In addition to increased rates of photosynthesis, asubstantial and significant increase in the growth of the

Table I. Photosynthetic parameters of wild-type and Rieske FeS ox lines determined from light response curves carried out at 2% [O2] (see Fig. 3, Aand B)

Statistical differences are shown in boldface (* P , 0.1; ** P , 0.05; and *** P , 0.01). Asat, Light-saturated rate of CO2 fixation. SE values areshown.

NameParameters

Light Fq9/Fm9 ETRII qP Asat

mmol m22 s21 mmol e2 m22 s21 mmol m22 s21

Wild type 400 0.343 6 0.013 62.3 6 0.96 0.58 6 0.02 14.7 6 0.351,000 0.124 6 0.002 54.4 6 0.91 0.23 6 0.01

9 400 0.380 6 0.004** 66.5 6 0.46** 0.64 6 0.00** 16.5 6 0.13**1,000 0.151 6 0.002*** 65.9 6 0.79*** 0.28 6 0.00**

10 400 0.369 6 0.009 65.9 6 0.68** 0.62 6 0.01 17.7 6 0.87***1,000 0.147 6 0.003*** 64.3 6 1.24*** 0.27 6 0.00*

11 400 0.370 6 0.018 66.4 6 3.73 0.63 6 0.02 18.4 6 0.44***1,000 0.156 6 0.006*** 70.3 6 2.47*** 0.29 6 0.00***

Table II. Maximum electron transport rate (Jmax) and maximumassimilation (Amax) in wild-type and Rieske FeS ox lines

Results were derived from the A/Ci response curves shown in Figure3C using the equations published by von Caemmerer and Farquhar(1981). Statistical differences are shown in boldface (* P , 0.1 and** P , 0.05). SE values are shown.

Name Parameters

Jmax

(mmol m22 s21)

Amax

(mmol m22 s21)

Wild type 181 6 11.6 24.4 6 2.319 210 6 10.2* 31.1 6 1.14**10 194 6 8.9 28.3 6 0.6711 216 6 11.9** 31.9 6 0.68**

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rosette area was observed in the Rieske FeS ox plants inthe early vegetative phase, which resulted in an approx-imately 30% to 70% increase in biomass yield in the dif-ferent lines. Importantly, seed yields in line 11, whichshowed the biggest increases in shoot biomass, also were

shown to be increased relative to thewild type. Given thatincreases in leaf area are evident in the Rieske ox plantsfrom early in development, the improvements in biomassare likely due to a combination of increased light captureand photosynthesis due to the greater leaf area.

Figure 4. Determination of the efficiency of electrontransport in leaves of young Rieske FeS ox plants.Wild-type (WT) and Rieske FeS ox plants were grownin controlled-environment conditions with a lightintensity of 130 mmol m22 s21 and an 8-h-light/16-h-dark cycle, and the redox statewas determined (27DAP)using a Dual-PAM instrument at a light intensity of220 mmol m22 s21. The data were obtained using fourindividual plants from Rieske FeS ox line 11 comparedwith the wild type (five plants). Significant differencesare indicated (*, P , 0.05). Error bars represent SE.

Figure 5. Growth analysis of wild-type (WT) andRieske FeS ox plants. Plants were grown at 130 mmolm22 s21 light intensity in short days (8 h of light/16 h ofdark). A, Appearance of plants at 10 and 18 DAP. B,Leaf area determined at 20 DAP. Significant differ-ences (*, P, 0.01 and **, P, 0.001) are indicated. C,Final biomass at 25 DAP. Results are representative ofsix to nine plants from each line. Increase over wildtype (%) is indicated as numbers on the histogram.Significant differences (P , 0.05) are represented byuppercase letters. Error bars represent SE.

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Rieske FeS ox Plants Have Increased Levels of Proteins inthe cyt b6f, PSI, and PSII Complexes

In keeping with our analysis of the electron transportprocesses in the Rieske FeS ox plants, increases in thelevels of cyt b6 and cyt f, core proteins of the cyt b6fcomplex, were evident. Furthermore, increases in thelevels of proteins in both PSII and PS1 and the d-subunitof the ATPase complex also were observed. This result

was unexpected, given that no changes in the compo-nents of PSII or PSI were observed in the Rieske FeSantisense plants. In contrast, in keeping with this study,the hcf mutant, in which the biogenesis of the cyt b6fcomplex was inhibited, also had a reduced accumula-tion of components of both PSI and PSII (Lennartz et al.,2001). In addition, a recent study reported increases incyt b6f protein levels in Arabidopsis plants grown undersquare-wave light compared with plants grown underfluctuating light, and these were accompanied by in-creased levels of PSII, PSI, and the d-subunit of theATPase proteins (Vialet-Chabrand et al., 2017). Takentogether, these results provide further demonstration ofthe ability of the thylakoid membrane to respond tochanges in the external and internal environments(Foyer et al., 2012). Although there are many examplesof coordination of the accumulation of photosyntheticcomplexes in the thylakoid membrane, the underlyingregulation of the synthesis and assembly of compo-nents of the thylakoid membrane, the factors deter-mining the accumulation of these complexes, is stillpoorly understood (Schöttler et al., 2015). Recently, anucleus-encoded chloroplast RNA-binding protein,photosystem biogenesis regulator1 (PBR1), was iden-tified and shown to be involved in the coordination ofthe biogenesis of the PSI, cyt b6f, and NDH complexes(Yang et al., 2016). It will be interesting to explore therole of PBR1 in the plants produced in this study, asthese are likely useful tools to investigate fundamentalquestions on factors controlling both the biogenesisand activities of the photosynthetic complexes in theelectron transport chain.

A recent study has shown that, for rice plants growingin elevated CO2, a small decrease in Rubisco proteinlevels resulted in an increase in the electron transportcomponents and an increase in biomass (Kanno et al.,2017). Given the increase in thylakoid protein composi-tion of the Rieske ox plants, it might be expected thatthere would be some tradeoff, and a reduction in otherproteins may occur to compensate. Although no com-prehensive analysis of proteins has been undertaken inthese plants, we found no evidence of changes in thelevels of Rubisco, which is a major N sink or FBPA andGDC. What is not yet clear is the impact of this manipu-lation on nitrogen use efficiency (NUE), and a full analysisof the performance of these plants under different N re-gimes will be needed to explore this question.

Overexpression of Rieske FeS Significantly Modifies thePigment Content of Leaves

In parallel with the increase in components of thethylakoidmembranes, plants with increased Rieske FeSprotein were found to have increases in the levels ofboth chlorophyll a and b and a small increase in thechlorophyll a/b ratio, from 2.96 to 3.12. The increases inchlorophyll a and b suggest a greater investment in bothlight capture and PSII reaction centers and would fitwith the increase in photosynthetic electron transport

Figure 6. Seed yield and vegetative biomass of wild-type (WT) andRieske FeS ox plants. Plants were grown at 130 mmol m22 s21 light in-tensity in short days (8 h of light/16 h of dark). A, Appearance of plants at38 DAP. B and C, Final biomass (B) and seed yield at harvest (52 DAP;C). Increase over the wild type (%) is indicated by numbers on thehistograms. Results are representative of six to nine plants from eachline. Significant differences (P , 0.05) are represented by uppercaseletters. Error bars represent SE.

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capacity in the Rieske FeS ox plants. In previous work,plants with reduced levels of the Rieske FeS protein had alower chlorophyll a/b ratio (Hurry et al., 1996; Price et al.,1998), which is the opposite of what was observed in theRieske FeS ox plants. In addition to increases in chloro-phyll, significant increases in the carotenoid pigments alsowere seen with b-carotene (+169%), violaxanthin (+59%),lutein (+75%), and neoxanthin (+37%). b-Carotene is acomponent of both the reaction centers (RC) and light-harvesting complex (Kamiya and Shen, 2003; Ferreiraet al., 2004; Loll et al., 2005; Litvin et al., 2008; Janik et al.,2016), and the increase in these pigments observed in the

Rieske FeS ox plants is in agreement with the investmentin both light harvesting and increasing RC efficiency. Wealso observed a significant decrease in the chlorophyll-to-carotenoid ratio in Rieske FeS ox plants, which wouldsuggest that there is a greater investment in photo-protection relative to the increased chlorophyll for lightharvesting. This increase in carotenoids relative tochlorophyll may contribute to the lower NPQ observedin Rieske FeS ox plants (Kaňa et al., 2016).

Lutein, neoxanthin, and violaxanthin are the mainxanthophyll pigment constituents of the largest light-harvesting pigment-protein complex of photosystem II

Figure 7. Pigment content in wild-type (WT) and Rieske FeS ox plants. Plants were grown at 130 mmol m22 s21 light intensity inshort days (8 h of light/16 h of dark). Two leaf discs, collected from two different leaves, were immersed inN,N-dimethylformamide(DMF) at 4°C for 48 h and separated by ultra-performance liquid chromatography. Results are represented as mg g21 fresh weight(FW). Statistical differences are shown by asterisks (*, P , 0.1; **, P , 0.05; and ***, P , 0.001). Error bars represent SE.

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(Thayer and Björkman, 1992; Ruban et al., 1994, 1996,1999; Ruban, 2012; Janik et al., 2016). Acidification ofthe thylakoid lumen as a result of electron transport(and driven in particular by the activities of the cyt b6fcomplex) is accompanied by the deepoxidation of viola-xanthin and an accumulation of zeaxanthin (Björkmanand Demmig-Adams, 1994; Müller et al., 2001; Ruban,2012) aswell as protonation of carboxylic acid residues ofthe PsbS protein associated with PSII antennae (Li et al.,2000, 2004). Protonation of PsbS and binding of zeaxan-thin increase NPQ and the thermal dissipation of exci-tation energy (Baker, 2008; Jahns and Holzwarth, 2012).However, we found that the increase in the level of theRieske FeS protein led to small but significantly lowersteady-state levels of NPQ. The absence of an increase inNPQ in the presence of significant increases in electrontransport rates suggests that the Rieske FeS ox plantsalso have increased rates of ATP synthesis. Althoughwe provide no direct support for this, we did observean increase in the level of the ATP synthase d-subunitprotein in the Rieske FeS ox plants. Further support forthis suggestion comes from earlier work on Rieske FeSantisense plants, which showed that the level of ATPsynthase and the transthylakoid pH gradient wereboth reduced (Price et al., 1995, 1998; Ruuska et al.,2000).

CONCLUSION

A number of studies have shown that increasingphotosynthesis through the manipulation of CO2 as-similation can improve growth (Miyagawa et al., 2001;Lefebvre et al., 2005; Rosenthal et al., 2011; Uematsuet al., 2012; Simkin et al., 2015, 2017). This work, to-getherwith a study inwhich cytochrome c6 from the redalga Porphyra was expressed in Arabidopsis (Chidaet al., 2007), provides direct evidence that there is alsoan opportunity to improve the efficiency of the electrontransfer chain. Here, we have shown not only thatoverexpression of the Rieske FeS protein resulted inincreases in cyt f and cyt b6 proteins of the cyt b6f com-plex but that components of PSI, PSII, and the ATPasealso were increased. Under the conditions of growthused in this study, these changes led to improvementsin photosynthesis and biomass obtained from theRieske ox plants relative to the wild type.

MATERIALS AND METHODS

Rieske FeS Protein of cyt b6f

The full-length coding sequence of the Rieske FeS protein of the cyt b6f(X64353) complex was amplified by reverse transcription-PCR using primersNtRieskeFeSF (59-caccATGGCTTCTTCTACTCTTTCTCCAG-39) and NtRieske-FeSR (59-CTAAGCCCACCATGGATCTTCACC-39). The resulting amplifiedproduct was cloned into pENTR/D (Invitrogen) to make pENTR-NtRieskeFeS,and the sequence was verified and found to be identical. The full-length cDNAwas introduced into the pGWB2 Gateway vector (Nakagawa et al., 2007;AB289765) by recombination from the pENTR/D vector tomake pGW-NtRieske(B2-NtRi). cDNAs are under transcriptional control of the 35S tobacco mosaic

virus promoter, which directs constitutive high-level transcription of the trans-gene, followed by the nos 39 terminator. Full details of the B2-NtRi constructassembly can be seen in Supplemental Figure S1.

Generation of Transgenic Plants

The recombinant plasmid B2-NtRi was introduced into wild-type Arabi-dopsis (Arabidopsis thaliana) by floral dipping (Clough and Bent, 1998) usingAgrobacterium tumefaciens GV3101. Positive transformants were regenerated onMurashige and Skoog medium containing kanamycin (50 mg L21) andhygromycin (20 mg L21). Kanamycin/hygromycin-resistant primary trans-formants (T1 generation) with established root systems were transferred to soiland allowed to self-fertilize.

Plant Growth Conditions

Wild-type T2 Arabidopsis plants resulting from self-fertilization of trans-genic plants were germinated in sterile agarmedium containingMurashige andSkoog salts, selected on kanamycin, and grown to seed in soil (Levington F2;Fisons), and lines of interest were identified by western blot and quantitativePCR. For experimental study, T3 progeny seeds from selected lines weregerminated on soil in controlled-environment chambers at an irradiance of130 mmol m22 s21 in an 8-h/16-h square-wave photoperiod with an air tem-perature of 22°C and a relative humidity of 60%. Plant position was ran-domized, and the position of the trays was rotated daily under the light. Leafareas were calculated from photographic images using ImageJ software(imagej.nih.gov/ij). Wild-type plants used in this study were a combinedgroup of the wild type and null segregants from the transgenic lines, verifiedby PCR for nonintegration of the transgene, as no significant differences ingrowth parameters were seen between them (Supplemental Fig. S2).

Protein Extraction and Immunoblotting

Four leaf discs (0.6 cm diameter) from two individual leaves were taken,immediately plunged into liquid N2, and subsequently stored at 280°C. Sam-ples were ground in liquid nitrogen, and protein quantificationwas determined(Harrison et al., 1998). Samples were loaded on an equal protein basis, sepa-rated using 12% (w/v) SDS-PAGE, transferred to a polyvinylidene difluoridemembrane, and probed using antibodies raised against the cyt b6f complexproteins cyt f (PetA; AS08306), cyt b6 (PetB; AS03034), Rieske FeS (PetC;AS08330), the PSI Lhca1 (AS01005) and PsaA (AS06172) proteins, the PSIIPsbA/D1 (AS01016) and PsbD/D2 (AS06146) proteins, the ATP synthase d-sub-unit (AS101591), and the Gly decarboxylase H-subunit (AS05074), all purchasedfrom Agrisera (via Newmarket Scientific). FBPA antibodies were raised against apeptide from a conserved region of the protein [C]-ASIGLENTEANRQAYR-amide(Cambridge Research Biochemicals; Simkin et al., 2015). Proteins were detectedusing horseradish peroxidase conjugated to the secondary antibody and ECLchemiluminescence detection reagent (Amersham). Proteins were quantifiedusing a Fusion FX Vilber Lourmat imager (Peqlab), as described previously(Vialet-Chabrand et al., 2017).

Chlorophyll Fluorescence Imaging

Chlorophyll fluorescence measurements were performed on 10-d-old Ara-bidopsis seedlings that had been grown in a controlled-environment chamber ata photosynthetic photon flux density (PPFD) of 130 mmol m22 s21 with ambientCO2 at 22°C. Images of the operating efficiency of PSII photochemistry (Fq9/Fm9)were taken at PPFDs of 310 and 600 mmol m22 s21 using a chlorophyll fluo-rescence imaging system (Technologica; Barbagallo et al., 2003; Baker andRosenqvist, 2004). Fq9/Fm9, was calculated from measurements of steady-statefluorescence in the light (F9), and maximum fluorescence in the light (Fm9) wasobtained after a saturating 800-ms pulse of 6,200 mmol m22 s21 PPFD using thefollowing equation: Fq9/Fm9 = (Fm9 2 F9)/Fm9 (Oxborough and Baker 1997a;Baker et al., 2001).

A/Ci Response Curves

The response ofA to Ci was measured using a portable gas-exchange system(CIRAS-1; PP Systems). Leaves were illuminated using a red-blue light sourceattached to the gas-exchange system, and light levels were maintained at sat-urating PPFD of 1,000 mmol m22 s21 with an integral light-emitting diode light

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source (PP Systems) for the duration of theA/Ci response curve. MeasurementsofAwere made at ambient CO2 concentration (Ca) of 400 mmolmol21, before Cawas decreased in a stepwisemanner to 300, 200, 150, 100, and 50mmolmol21 beforereturning to the initial value and increased to 500, 600, 700, 800, 900, 1,000, 1,100,and 1,200 mmol mol21. Leaf temperature and vapor pressure deficit weremaintained at 22°C and 1 6 0.2 kPa, respectively. The maximum rate ofRubisco and the maximum rate of electron transport for ribulose 1,5-bisphosphate regeneration were determined and standardized to a leaf tem-perature of 25°C based on equations from Bernacchi et al. (2001) andMcMurtrie and Wang (1993), respectively.

Photosynthetic Capacity

Photosynthesis as a function of PPFD (A/Q response curves) was measuredusing the 6400XT portable gas-exchange system (Li-Cor). Cuvette conditionswere maintained at a leaf temperature of 22°C, relative humidity of 50% to 60%,and ambient growth CO2 concentration of 400 mmol mol21 for plants grown inambient conditions. Leaves were initially stabilized at saturating irradiance of1,000 mmol m22 s21, after which A and stomatal conductance were measured atthe following PPFD levels: 0, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 800,and 1,000 mmol m22 s21. Measurements were recorded after A reached a newsteady state (123 min) and before stomatal conductance changed to the newlight levels. A/Q analyses were performed at 21% and 2% [O2].

PSI and PSII Quantum Efficiency

ThephotochemicalquantumefficiencyofPSII andPSI in transgenicandwild-type plants was measured following a dark-light induction transition using aDual-PAM-100 instrument (Walz) with a DUAL-DR measuring head. Plantsweredark adapted for 20minbefore placing in the instrument. Followingadark-adapted measurement, plants were illuminated with 220 mmol m22 s21 PPFD.The maximum quantum yield of PSII was measured following a saturatingpulse of light for 600 ms at an intensity of 6,200 mmol m22 s21. The PSII op-erating efficiency was determined as described by the routines above. PSIquantum efficiency was measured as an absorption change of P700 before andafter a saturating pulse of 6,200 mmol m22 s21 for 300 ms (which fully oxidizesP700) in the presence of far-red light with a far-red preillumination of 10 s.Both measurements were recorded every 1 min for 5 min. qp or Fv9/Fm9 wascalculated from measurements of steady-state fluorescence in the light (F9)and maximum fluorescence in the light (Fm9), while minimal fluorescence inthe light (Fo9) was calculated following the equation of Oxborough and Baker(1997b). The fraction of open PSII centers (qL) was calculated from qp 3 Fo9/F(Baker, 2008).

Pigment Extraction and HPLC Analysis

Chlorophylls and carotenoids were extracted using DMF as described pre-viously (Inskeep and Bloom, 1985), which was shown subsequently to suppresschlorophyllide formation in Arabidopsis leaves (Hu et al., 2013). Briefly, twoleaf discs collected from two different leaves were immersed in DMF at 4°C for48 h and separated by ultra-performance liquid chromatography as describedby Zapata et al. (2000).

Leaf Thickness

Leaves of equivalent developmental stage were collected from plants after28 d of growth. Strips were cut from the center of the leaf, avoiding themidvein,preserved in 5%glutaraldehyde, stored at 4°C for 24 h, followed by dehydrationin sequential ethanol solutions of 20%, 40%, 80%, and 100% (v/v). The sampleswere placed in LR White acrylic resin (Sigma-Aldrich), refrigerated for 24 h,embedded in capsules, and placed at 60°C for 24 h. Sections (0.5 mm) were cutusing a Reichert-Jung Ultracut microtome (Ametek), fixed, stained, and viewedwith a light microscope (López-Juez et al., 1998). Leaf thickness was determinedby measuring leaves from two to three plants from lines 9 and 11 comparedwith leaves from four wild-type plants.

Statistical Analysis

All statistical analyses were done by comparing ANOVA using Sys-stat(University of Essex). The differences between means were tested using theposthoc Tukey’s test (SPSS).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession number X64353.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic representation of the Rieske FeS over-expression vector pGWRi used to transform Arabidopsis.

Supplemental Figure S2. Determination of photosynthetic efficiency andleaf area of the wild type versus azygous segregating controls usingchlorophyll fluorescence imaging.

Supplemental Figure S3. Immunoblot analysis of wild-type and RieskeFeS ox proteins.

Supplemental Figure S4. Light response curves of the Rieske FeS plants at21% [O2].

Supplemental Figure S5. Determination of the efficiency of electron trans-port in leaves of mature Rieske FeS ox plants.

Supplemental Figure S6. Determination of the efficiency of electron trans-port in leaves of young and mature Rieske FeS ox plants.

Supplemental Table S1. Physiological parameters of Rieske FeS ox plantscompared with the wild type.

ACKNOWLEDGMENTS

We thank James E. Fox and Philip A. Davey for help with pigment analysis,Silvere Vialet-Chabrand for help in drafting revisions of the article, and Elena A.Pelech for help with Dual-PAM measurements.

Received May 9, 2017; accepted July 27, 2017; published July 28, 2017.

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